The purinergic P2U or nucleotide receptor is an integral part of the plasmalemma of various mammalian cell types. The PU2 receptor described in this application is most similar to a G-protein coupled surface receptor from rat. These receptors are associated with cells such as neutrophils, endothelial cells, and fibroblasts in the immune, neural, muscular, pulmonary and vascular systems. P2U receptors stimulate phosphoinositide metabolism and the release of intracellular Ca++ in the presence of extracellular nucleotides, particularly UTP or ATP. In macrophages, Mg++ inhibits the response of P2U to ATP (Alonso-Torre S R and A Trautmann (1994) J Biol Chem 268:18640-47); and in lung epithelial cells, stimulation of the P2U receptor by nucleotides modulates chloride secretion. P2 receptors have a very low affinity for adenosine and are not activated by the methylxanthine antagonists, caffeine and theophylline.
The P2U receptor is in the P2 receptor family for which the common structural features have been described: 1) seven hydrophobic domains, 2) consensus N-linked glycosylation sequences near the amino terminus, 3) a number of residues common to G-protein coupled receptors (asn51, asp79, cys106, and cys183), and 4) potential phosphorylation sites in the third intracellular and carboxyterminal domains (Parr C E et al (1994) Proc Natl Acad Sci 91:3275-79).
In addition to P2U, there are four other P2 receptor subtypes. The P2X receptor mediates smooth muscle response following sympathetic nerve stimulation and contains an intrinsic cation channel. The P2Y receptor is found in smooth muscle and vascular tissue where it induces vasodilation in response to nitric oxide. The P2Z receptor is found primarily on mast or other immune cells, and when activated by ATP, it appears to cause cell permeabilization. The P2T receptor, which is only found on platelets, inhibits adenylate cyclase and stimulates the release of intracellular calcium ions. In contrast, P1 receptors are stimulated by adenosine rather than nucleotides.
The G-protein coupled receptors (T7G) characteristically contain seven hydrophobic domains which span the plasma membrane and form a bundle of antiparallel xcex1 helices. These transmembrane segments are designated by roman numerals and account for many of the structural and functional features of the receptor. In most cases, the bundle of helices forms a binding pocket; however, the binding site for bulky molecules includes the extracellular N-terminal segment or one or more of the three extracellular loops. Binding may also alter the receptor""s intracellular configuration (Watson S and Arkinstall S (1994) The G-Protein Linked Receptor Facts Book, Academic Press, San Diego, Calif.).
The activated receptor interacts with an intracellular G-protein complex which mediates further intracellular signalling activities, generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate, or ion channel proteins. Coupling to G-proteins involves a variable sequence in the C-terminal 10-20 amino acids of the third internal loop between the transmembrane segments V and VI and the intracellular segment immediately C-terminal to transmembrane segment VII. Interaction with Gq also requires the N-terminal 10-20 amino acids of the third internal
Both structural and functional features of T7Gs allow their classification into five categories: xcex2-type, muscarinic-type, neurokinin-type, nonneurokinin-type, and miscellaneous (Bolander F F (1994) Molecular Endocrinology, Academic Press, San Diego, Calif.); each of which are discussed below. P2U is a xcex2-type receptor and has structural features shared with xcex2-adrenergic, xcex1-adrenergic, histamine, dopamine, and serotonin receptors. These receptors have a short N-terminus with two conserved N-glycosylation sites, a moderately short third internal loop, and a long C-terminus containing a Ser/Thr-rich region. All adrenergic receptors elevate cAMP or intracellular calcium.
The novel purinergic receptor which is the subject of this patent application was identified among the cDNAs derived from a placental library. Incyte Clone 179696 is a novel homolog of RNU09402, a G-protein coupled surface receptor from rat (Rice WR et al (1995) Am J Respir Cell Molec Biol 12:27-32). Purinergic receptors of the: placenta are likely found on immune or vascular cells and appear to play an important role in signal transduction and other specialized functions of the placenta as briefly described below.
Placenta
The placenta is a thickened discoid temporary organ that acts as the site of interchange of substances between the maternal and fetal bloodstreams. Such substances include oxygen, nutrients, hormones, excretory products, humoral antibodies (immunoglobulin G, IgG), drugs, viruses, or any other chemical or infectious agent that may be present in the maternal circulation.
The placenta consists of a fetal part derived from the chorion, one of the extraembryonic surrounding membranes of the conceptus and of a maternal part (decidua basalis) derived from the region of endometrium that underlies the implantation site. The placenta is thus the only organ composed of cells derived from two individuals. The boundary between maternal and fetal tissues is marked by extracellular products of necrosis referred to as fibrinoid. The anatomy of the human placenta is discussed in detail in Benirschke and Kaufmann, (1992) Pathology of the Human Placenta, Springer-Verlag, New York City, pp 542-635.
Development
The late blastocyst consists of an inner cell mass that gives rise to the embryo and an outer, single layer of trophoblast cells that encloses the blastocyst cavity. Following implantation, trophoblasts become highly invasive, erode and attach to the secretory endometrium. This invasive process involves matrix-degrading metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), adhesion receptors and their extracellular ligands, and the class I human leukocyte antigen-G (HLA-G) molecule. The invasive process is reviewed in Fisher and Damsky (1993 Semin Cell Biol 4(3):183-188) and in Graham and Lala (1992 Biochem Cell Biol 70:867-874).
Trophoblasts give rise to two layers. The inner layer is composed of individual cells, cytotrophoblasts, which have high proliferative potential. The outer layer is composed of syncytial cells, syncytiotrophoblasts, which invade the endometrium and become surrounded by cavernous spaces (lacunae) filled with maternal blood. Finger-like extensions of the cytotrophoblasts grow into these protrusions and act as primary placental villi. The capillaries found in this tissue are a part of the embryonic circulation. Tufted extensions of part of the chorion or chorionic villi are associated with the decidua basalis and develop into the large, elaborately branched outgrowths of the villous chorion. The syncytiotrophoblasts remain until the end of pregnancy, but by the fifth month of gestation, most of the cytotrophoblasts begin to fuse with the syncytiotrophoblast. The few remaining cytotrophoblasts form a discontinuous basal layer.
Chorion
The chorion or fetal part of the placenta has a chorionic plate at the point where the chorionic villi arise. The finger-like villi extend into the endometrial lacuna which are filled with maternal blood released under pressure from the endometrial spiral arteries. A connective tissue core in which the fetal blood vessels develop is derived from extraembryonic mesenchyme surrounded by syncytiotrophoblast and cytotrophoblast cell layers.
During pregnancy, surface area of the villi increases dramatically. The surfaces of the villi are active in the exchange of substances between fetal and maternal circulatory systems. Receptors within the apical microvilli facilitate transport of glucose, amino acids, and IgG from mother to fetus. The mechanism for IgG movement is similar to that of IgA across epithelia. The transport of various materials, particularly nutrients, by the placenta is reviewed in Smith et al (1992 Ann Rev Nutrition 12:183-206) and Schneider (1991 Reprod Fertil Dev 3:345-353). The placenta is more than a simple conduit for nutrients; it engages in considerable metabolic activity contributing to the quality and quantity of nutrients supplied to the fetus (cf. Hay (1991) Diabetes 40S:44-50).
Although the villi express foreign paternal as well as maternal antigens and a maternal immune response would be expected against the fetal xe2x80x9callograftxe2x80x9d, the fetus is not usually rejected. The type of Fetal factors such as major histocompatibility complex (MHC) I (but not MHC II) and low antigen density and maternal response (suppressor cells and molecules) all contribute to a complex and unique tolerance. The absence of MHC II may be particularly significant, since MHC II has been implicated in the rejection of organ allografts.
Decidua Basalis
The function of the endometrium is to support the implantation and development of the embryo. During each menstrual cycle, the most superficial layer or functionalis, undergoes dramatic changes in preparation for these events. During proliferative phase in the first half of the cycle, rising estrogen levels stimulate the division of epithelial and stromal cells in the functionalis. The uterine lining is ready by the time of ovulation at day 14.
During the secretory phase in the second half of the cycle, endometrial cells differentiate in response to rising levels of progesterone. Beginning as early as day 15, glycogen appears in the basal region of the epithelial cells and displaces the nuclei. By day 18, the glycogen is dispersed, the nuclei have returned to a basal portion of the cell, Golgi are prominent apically, and secretion is maximal. Concurrently, the nuclear envelope indents to form a channel system associated with the nucleolus. This system is believed to facilitate a rapid transfer of ribosomal components between the nucleus and the cytoplasm. Uterine secretions contain significant amounts of glucose and specific glycoproteins such as PP14 which may confer immunosuppression in preparation for contact with the xe2x80x9cforeignxe2x80x9d embryo.
Implantation induces a decidual response that is characterized by pronounced changes in the endometrial stroma. Fibroblast-like cells transform into large, active decidual cells that become an important component of the decidua basalis. Predecidual cells, which appear in the endometrial stroma during the fourth week of every menstrual cycle, form a cuff around small vessels in the stroma. The vessels become more permeable as menstruation or placental development approaches.
The predecidual cells appear to limit embryo invasion, play a role in embryo nutrition, and protect fetal tissue from rejection. These cells produce prolactin (and possibly relaxin), secrete prostaglandins, and have receptors for both estrogen and progesterone. The effects of estrogen and progesterone on the endometrium, both during the cycle and following implantation, are complemented and implemented by a variety of growth factors. Insulin-like growth factors (IGFs) have a major role in the stimulation of endometrial cell division. With rising levels of progesterone after ovulation, IGF-binding proteins, including the placental protein PP12 synthesized by the predecidual cells, are secreted. IGF-binding proteins reduce the availability of IGFs and thus play a role in the shift from a proliferative to a secretory endometrium.
The decidua basalis supplies arterial blood to and receives venous blood from the lacunae situated between the villi. Although the maternal blood vessels are open during implantation, the fetal vessels remain intact. Fetal and maternal blood do not mix, except on rare occasions at the end of pregnancy. During this period when the cytotrophoblast is no longer continuous and the capillaries of the villi are very close to the surface, a very slight exchange of blood may occur. At that time, the walls of the fetal capillaries are separated from the maternal blood only by the syncytiotrophoblast.
During pregnancy, cells from the connective tissue stroma of the decidua basalis and a lesser number of cells from the decidua parietalis and decidua capsularis form decidual cells. These large, slightly basophilic cells have many profiles of rough endoplasmic reticulum, long mitochondria, and membrane-limited granules contained in club-shaped projections of the cell surface. Decidual cells are more numerous during the first half of pregnancy, contain a nucleus with a prominent nucleolus, and secrete prolactin which is similar to pituitary prolactin.
At the end of a full-term pregnancy, the placenta has the shape of a thick disk. The umbilical cord usually arises from the center of the placenta and connects the circulation of the fetus with the fetal placental circulation. Fetal venous blood reaches the placenta through the two umbilical arteries which branch and ultimately give rise to the vessels of the chorionic villi. In these villi, the fetal blood receives oxygen, loses its CO2 and returns to the fetus through the umbilical vein. Although the chorionic villi are submerged in maternal blood, the fetal placental blood is isolated by the structures that form the placental barrierxe2x80x94the endothelium and basal lamina of the fetal capillaries; the mesenchyme in the villus interior; the basal lamina of the trophoblast; the cytotrophoblast, during the first half of pregnancy; and the syncytiotrophoblast.
The placenta is permeable to several substances and normally transfers oxygen, water, electrolytes, carbohydrates, lipids, proteins, vitamins, hormones, antibodies, and some drugs from the maternal to the fetal circulation. Carbon dioxide, water, hormones, and residual products of metabolism are transferred from fetal blood to maternal blood. The complexity of this bidirectional transport reflects the function of the placental layers as the equivalent of three organ systemsxe2x80x94respiratory, gastrointestinal, and urinary. The mechanism of transport is extremely varied, ranging from simple diffusion of gases to many types of receptor-mediated transport including the active transport of amino acids and a special shuttle mechanism for IgG. IgG is the only immunoglobulin which crosses the placental barrier, enters fetal circulation, and protects the newborn against infection. Makiya and Stignrand (1992 Clin Chem 38:2543-45) suggest that placental alkaline phosphatase binds the Fc portion of IgG and acts as the placental IgG receptor.
Maternal Immunologic Tolerance of Fetal Tissue
Villi expressing foreign (paternal) antigens are exposed directly to maternal blood. Even though a maternal immune response occurs, fetal tissue is not typically rejected. Low expression of MHC I, absence of MHC II, and suppression of maternal response contribute to this unique tolerance. The trophoblast which is the true allograft and comes in contact with maternal blood, does not express classical MHC antigens. Occasionally, maternal IgG may harm the fetus relative to Rhesus factor (Rh) or maternal immune thrombocytopenic purpura.
An understanding of how the trophoblast/fetus escapes rejection might allow development of rational strategies for combating pregnancy disorders, such as preeclampsia or intrauterine growth retardation, having an immunological basis. The fetal-maternal immune interaction is reviewed in Herrera-Gonzalez and Dresser (1993 Dev Comp Immunol 17:1-18).
Placental Hormones
Soon after implantation, fetal villi begin to control maternal physiology creating an optimal environment for fetal development. Immediately after implantation, the syncytiotrophoblast synthesizes human chorionic gonadotropin (HCG), a glycoprotein hormone that mimics the effects of luteinizing hormone (LH) through the first few months of gestation. HCG has a subunit identical to that of LH and follicle-stimulating hormone (FSH). LH acts on and maintains the corpus luteum by stimulating estrogen and progesterone synthesis.
Beginning at about eight weeks into gestation, the syncytiotrophoblast assumes the role of the corpus luteum and begins to secrete estrogen and progesterone. The steroid hormones progesterone and estrogen are made by both kinds of trophoblast, but estrogen production requires the metabolic cooperation of the fetal adrenal cortex and liver. The syncytiotrophoblast which continues to produce these hormones throughout gestation utilizes both maternal and fetal androgen precursors to form estrogens and massive amounts are released into the maternal bloodstream.
Placental progesterone is synthesized from cholesterol obtained primarily from circulating low-density lipoprotein (LDL). Membranes of the microvilli provide surface area for LDL receptors. LDL is initially shuttled into lysosomes and cholesterol is released by the action of acid hydrolases. Then the cholesterol is transported to mitochondria where it is acted upon by enzyme complexes within the tubular cristae.
The syncytiotrophoblast is also the chief source of human chorionic somatomammotropin (HCS), a glycoprotein hormone with both lactogenic and growth-promoting activity. HCS is similar to growth hormone and has effects on maternal carbohydrate, fat, and protein metabolism. As maternal utilization of fatty acids increases, available glucose is reserved for the fetus. HCS has its major effect, in conjunction with prolactin, on development of the mammary gland.
Cytotrophoblasts produce significant amounts of platelet-derived growth factor-beta (PDGF-xcex2) as well as the PDGF-xcex1 and -xcex2 receptors (Holmgren et al (1992) Growth Factors 6:219-231). PDGF may play a role in cytotrophoblast proliferation. The action of various cytokines on the placenta is reviewed in Mitchell et al (1993 Placenta 14:249-275) and Rutanen (1993 Ann Med 25:343-347).
Pathology of the Placenta
Preeclampsia, now referred to as xe2x80x9cpregnancy-induced hypertensionxe2x80x9d (PIH), deserves special note. Common in pregnancy, preeclampsia is characterized by sudden development of hypertension, edema, and proteinuria. More severe toxemia or eclampsia includes convulsions and coma which may jeopardize both mother and fetus. The pathological changes of the placenta found in PIH are decidual arteriolopathy, infarcts, abruptio placenta, and Tenney-Parker changes.
The principal cause of preeclampsia is still unknown although it is certain that the disease relates to the presence of placental tissue, since the delivery of the placenta (or hydatidiform mole) ends the disease process. An obliterative thickening of arterial walls and a reduced number of small arteries in the villi have been observed and may explain the increase in vascular resistance in PIH. Another cause of uneven blood flow may be vasoconstriction. While the blood levels of the vasoconstrictor, angiotensin II, are not increased, uterine vascular responsiveness is greatly increased. Vasoconstriction may be induced by a reduction of unopposed thromboxane and angiotensin II. Reduced oxygen tension in the maternal blood supplied to the intervillous lacunae may also play a role.
Many types of infections by viruses, bacteria, mycoplasmas, or parasites cause pathological changes in the placenta. Infections may ascend from the endocervical canal, or they may reach the placenta through the maternal blood. Rarely are they acquired by amniocentesis, chorionic villus sampling, amnioscopy, percutaneous umbilical blood sampling, or intrauterine fetal transfusions. Some infections cause gross and microscopic changes of the placenta, while others leave few characteristic or specifically recognizable traces.
Other disorders of the placenta include, but are not limited to, abruptio placentae;
placenta previa; placental or maternal floor infarction; placenta accreta, increta, and percreta; extrachorial placentas; chorangioma; chorangiosis; chronic villitis; placental villous edema; widespread fibrosis of the terminal villi; intervillous thrombi; hemorrhagic endovasculitis; erythroblastosis fetalis; and nonimmune fetal hydrops. The pathology of the human placenta and decidua is discussed in Benirschke and Kaufmann, (1992) Pathology of the Human Placenta, Springer-Verlag, New York City pp. 542-635, and in Naeye (1992), Disorders of the Placenta, Fetus, and Neonate: Diagnosis and Clinical Significance, Mosby Year Book, St. Louis Mo.
The subject invention provides a unique nucleotide sequence which encodes a novel human purinergic P2U receptor (PNR). Incyte Clone No 179696 was used to identify and clone the full length cDNA (pnr) from the placenta cDNA library.
The invention also comprises the use of this PNR or its variants to intercede in physiologic or pathologic conditions and include diagnosis or therapy of activated or inflamed cells and/or tissues with pnr nucleic acids, fragments or oligomers thereof. Aspects of the invention include the antisense DNA of pnr; cloning or expression vectors containing pnr; host cells or organisms transformed with expression vectors containing pnr; a method for the production and recovery of purified PNR from host cells; purified protein, PNR, which can be used to generate antibodies for diagnosis or therapy of activated or inflamed cells and/or tissues.